Toxicology and safety study of L‐tryptophan and its impurities for use in broiler feed

L‐tryptophan has been utilized as a feed additive in animal nutrition to improve growth performance, as well as a dietary supplement to alleviate various emotional symptoms in humans. Despite its benefits, concerns regarding its safety arose following the outbreak of eosinophilia‐myalgia syndrome (EMS) among individuals who consumed L‐tryptophan. The causative material of EMS was determined to be not L‐tryptophan itself, but rather L‐tryptophan impurities resulting from a specific manufacturing process. To investigate the effect of L‐tryptophan and its impurities on humans who consume meat products derived from animals that were fed L‐tryptophan and its impurities, an animal study involving broiler chickens was conducted. The animals in test groups were fed diet containing 0.065%–0.073% of L‐tryptophan for 27 days. This study aimed to observe the occurrence of toxicological or EMS‐related symptoms and analyze the residues of L‐tryptophan impurities in meat products. The results indicated that there was no evidence of adverse effects associated with the test substance in the investigated parameters. Furthermore, most of the consumed EMS‐causing L‐tryptophan impurities did not remain in the meat of broiler chickens. Thus, this study demonstrated the safety of L‐tryptophan and some of its impurities as a feed additive.

In the late 1980s, an outbreak of eosinophilia-myalgia syndrome (EMS) cases emerged, which was associated with the consumption of L-tryptophan as a dietary supplement.Some individuals who consumed L-tryptophan experienced symptoms of EMS, including myalgia, muscle weakness, skin rashes, and abdominal pain (NORD, 2017).
The most prominent features of EMS are abnormal symptoms and inflammatory lesions on the skin, lungs, and muscles.Several case studies have shown that thick dermis and fascia on the skin, painful muscle spasms, and myopathy are frequently observed in EMS patients (Allen & Varga, 2014;Varga et al., 1993).Along with these systemic symptoms, an excessive increase in eosinophils is also noted in hematological tests.Also, it was reported that cytokines such as interleukins (ILs), granulocyte-monocyte colony-stimulating factor (GM-CSF), and transforming growth factors (TGFs) could potentially contribute to the progression of EMS (Allen et al., 2011;Owen et al., 1990;Varga et al., 1993).
There have been several studies suggesting that certain impurities in L-tryptophan are responsible for causing EMS, as well as the symptoms observed in EMS patients who have consumed tryptophan containing these impurities as a dietary supplement.L-tryptophan is also an essential amino acid for animals and should be included in their diets; however, there are not many studies on the safety of L-tryptophan containing these impurities in animals.Our previous study demonstrated the safety of L-tryptophan in rodents by assessing general toxicity and the occurrence of EMS-related symptoms from tissue lesions and immunological parameters reportedly associated with EMS (Ko et al., 2022).However, the safety implications for humans consuming meat products obtained from animals fed with L-tryptophan or L-tryptophan impurities have not been reported.Therefore, this study aimed to evaluate the safety of L-tryptophan and its impurities in broiler chickens by analyzing various toxicological and EMS-related parameters.Additionally, the residues of L-tryptophan impurities were examined to evaluate the potential human intake of these impurities through the consumption of broiler chickens.

| Test substances
Three types of L-tryptophan were used in this study: highly purified L-tryptophan, dried L-tryptophan fermentation product, and dried L-tryptophan fermentation product with additional impurities (Table 1).The dried L-tryptophan fermentation product consisted of L-tryptophan as the active substance and other components derived from the biomass of the fermentation broth.Since both the highly purified L-tryptophan and the dried L-tryptophan fermentation product contained HTP, IMT, MTCA, and PIC, these impurities were added to the dried L-tryptophan fermentation product to create a sample with a high level of L-tryptophan impurities.All test substances were produced through the fermentation of bioengineered Corynebacterium glutamicum (CJ CheilJedang, Suwon, Korea).
T A B L E 1 Composition of L-tryptophan and its impurities in the test substances.

| Acclimation and grouping of animals
Upon receipt, male and female broiler chickens were 1 day old with a mean body weight of 45 g.Following a 7-day acclimation period with a commercial starter diet, all broiler chickens reached the desired body weight (206 ± 0.2 g) by the eight day.They were then randomly assigned into four groups: Group 1 (G1, basal feed), Group 2 (G2, L-tryptophan), Group 3 (G3, dried L-tryptophan fermentation product), and Group 4 (G4, dried L-tryptophan fermentation product with additional impurities).
The diet was fed in two phases, with a grower diet provided for 8-to 21-day-old chickens and a finisher diet provided for 22-to 34-day-old chickens.Test substances were added to the diet in mash form for each treatment group.The composition of the diets are provided in the Supporting Information (Table S1).

| Clinical observations
All animals were visually monitored twice a day throughout the administration period to examine their health or response to treatment.Clinical signs such as mortality, morbidity, general appearance, and behavioral changes were examined.

| Body weight
All animals were weighed on the day before the commencement of test substance administration (Day 1), as well as on Days 7, 21, and 34 during the administration period, and before necropsy.

| Hematologic examination and clinical biochemistry
Following anesthesia with carbon dioxide, whole blood was collected from the heart and transferred to dipotassium ethylene diamine tetraacetic acid-coated tubes (K 2 EDTA, BD Bioscience, San Jose, CA, USA) and sodium citrated tubes (V-Tube, AB Medical, Gwangju, Korea) for hematological and coagulation tests, respectively.The blood samples were analyzed using a hematology system (Hemavet 950FS The whole blood samples remaining after the hematology and coagulation tests were collected into serum separation tubes (SST tubes, BD Bioscience).These tubes were stored at room temperature for at least 30 min and then centrifuged at 3000 rpm at 4 C for 10 min to separate the serum.The serum samples were subsequently analyzed using a biochemistry analyzer (HITACHI 7180, Hitachi Co., Ltd, Tokyo, Japan) to assess the levels of the following parameters: albumin, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transpeptidase, creatinine, blood urea nitrogen, total cholesterol, and glucose.

| Necropsy and organ weights
Following blood sampling conducted under deep anesthesia induced by carbon dioxide, the animals were euthanized via exsanguination through the aorta.Each animal was carefully observed for external abnormalities.Organs were removed and examined after the examination of any abnormalities in the abdominal, thoracic, and cranial cavities.The following organs and tissues were collected during necropsy and fixed in 10% neutral-buffered formalin: liver with gall bladder, kidneys, heart/aorta, lung/bronchus, spleen, thymus, pancreas, skeletal muscles from the breast and thigh, bursa of Fabricius, and skin.Following the necropsy, the absolute and relative (organ-to-body weight ratio) weights of the liver, kidneys, spleen, heart, and thymus were measured.For the kidneys, the combined weight of the left and right kidneys was calculated.

| Histopathological examination
Histopathological examinations were performed on tissues collected during necropsy for all animals.The obtained tissues were stored in 10% neutral-buffered formalin for over 24 h.Tissues were then routinely processed to obtain stained tissue samples for histopathology.
First, the formalin-fixed tissues were embedded in paraffin to create paraffin blocks, which were then sectioned using a rotary microtome (RM2235, Leica, Wetzlar, Germany).The resulting sections were automatically stained with hematoxylin and eosin using an autostainer (ST5010, Leica).Once the stained tissue was obtained, a microscopic examination was conducted.

| Statistical analyses
The body weight, organ weight, and data derived from hematology, coagulation, and clinical biochemistry tests were analyzed using IBM Statistical Package for the Social Sciences (SPSS) Statistics for Windows, Version 23.0 (IBM Corp., Armonk, NY, USA).The homogeneity of the group variances was evaluated using Levene's test, and a one-way analysis of variance (ANOVA) was employed to evaluate significant differences.Scheffé's multiple comparison test was conducted as a post hoc test when both homogeneity of variance and significance of difference were demonstrated.In the absence of homogeneous variation, Dunnett's T3 test was performed.For immunological analysis, one-way ANOVA followed by Bonferroni's test was conducted to evaluate significant differences using Statistical Analysis Software Version 9.4 (SAS Institute Inc., Cary, NC, USA).P values <0.05 were considered statistically significant.

| Analysis of L-tryptophan impurities
The chicken muscle samples from the thigh and breast were homogenized by grinding using a blender.One gram of the homogenized samples was placed into a conical tube, and 10 mL of 80% methanol was added.Then the mixture was shaken and sonicated for 1 h.After sonication, the samples were centrifuged at 15,000 rpm for 10 min, and the supernatants were filtered using a 0.2-μm polytetrafluoroethylene (PTFE) syringe filter.
The source parameters of optimized mass spectrometry were as follows: positive ion mode with 3500 V, sheath gas at 50 Arb, aux gas at 10 Arb, sweep gas at 1 Arb, ion transfer tube temperature set to 325 C, and vaporizer temperature set to 350 C. Data analysis was performed using TraceFinder 4.1 software (Thermo Scientific, USA).
Selected reaction monitoring transitions were monitored to quantify the analytes.
T A B L E 2 Mean body weight and body weight gain in male and female broiler chickens fed test substances for 27 days.T A B L E 3 Mean hematologic and coagulation parameters in male and female broiler chickens fed test substances for 27 days.Note: G1, basal feed; G2, L-tryptophan; G3, dried L-tryptophan fermentation product; G4, dried L-tryptophan fermentation product with additional impurities.Note: G1, basal feed; G2, L-tryptophan; G3, dried L-tryptophan fermentation product; G4, dried L-tryptophan fermentation product with additional impurities.3 | RESULTS

| Body weight changes and clinical observations
Mean body weight and body weight gain for 27 days in all animals are summarized in Table 2.The mean body weight of Group 1 was significantly lower compared to the other groups, and there was also a significant difference in the extent of body weight gain between Group 1 and the other groups.There were no mortalities or clinical signs observed during the administration period in any of the animals (data not shown).

| Hematology and clinical biochemistry
Table 3 summarizes the analytical data for hematology and coagulation assays conducted on all animals that were fed the test substance for 27 days.Those assays were conducted on all animals using an auto blood analyzer and coagulation analyzer.Hematological examination revealed a significant increase in the ratios of eosinophils and basophils in male broiler chickens from Groups 3 and 4 relative to Group 1. Conversely, female broiler chickens from Groups 3 and 4 exhibited statistically significant decreases in leukocytes and differential leukocyte counts (neutrophils, lymphocytes, monocytes, eosinophils, and basophils) compared with Group 1, but the magnitude of the changes was small.In the blood coagulation test, prolongations of prothrombin time and activated partial thromboplastin time were observed in Groups 3 and 4 in both male and female broiler chickens; however, no statistical significance was observed for these changes when compared to Group 1.
In terms of clinical biochemistry, serum aspartate aminotransferase and alanine aminotransferase levels were slightly elevated in male broiler chickens from Group 2 compared with those in Group 1, although no statistical differences were observed (Table 4).There were no other significant intergroup differences observed in both male and female broiler chickens.

| Immunological analysis
The results of the immunological analysis by ELISA are shown in Figure 1.The level of TGF-β1 was not graphed due to its low numerical value.A significant decrease in IL-4 was observed in male broiler chickens in Group 3 compared with those in Group 2. However, there were no significant intergroup differences observed in female broiler chickens.In terms of TGF-β2, female broiler chickens in Group 4 showed significantly higher values compared with those in Group 1. Regarding TGF-β3, significant differences were observed between Groups 2 and 4 in both sexes, as well as between Groups 3 and 4 in male broiler chickens.In terms of IL-5 and IL-6, no significant intergroup differences were observed in both sexes.
T A B L E 5 Mean relative organ weights in male and female broiler chickens fed test substances for 27 days.Note: G1, basal feed; G2, L-tryptophan; G3, dried L-tryptophan fermentation product; G4, dried L-tryptophan fermentation product with additional impurities.
T A B L E 6 Histopathological analysis of male and female broiler chickens fed test substances for 27 days.

| Gross necropsy and organ weights
No remarkable gross lesions were found in any of the animals during necropsy (data not shown).Figure 2 and Table 5 present the relative organ weights of all animals that were fed the test substance for 27 days.During the dosing period, no statistically significant difference was observed in the relative organ weights in all animals.

| Histopathological examination
Table 6 shows the results of the histopathological examination conducted on the liver, lung, heart, kidney, pancreas, thymus, bursa of Fabricius, skeletal muscles of breast and thigh, and skin of all animals.In the liver of all test groups, most of the animals exhibited inflammatory cell infiltration, extramedullary hematopoiesis, and hepatocyte degeneration.Occasional findings in the heart, lung, and kidney were observed in all test animals.There were no significant differences in the incidence and severity of lesions observed in the liver, heart, lung, and kidney among the four groups.Notably, there was a high frequency of muscle fiber degeneration and necrosis observed in the breast muscle (Figure 3), while sporadic instances of erosion or necrosis were also observed in the epidermis of the skin.
No specific findings were observed in the spleen.

| Residue of L-tryptophan impurities in chicken muscle
To assess the extent to which ingested L-tryptophan impurities were incorporated into animal tissues, muscle samples from the thigh and breast were collected and analyzed for residual L-tryptophan impurities using LC-MS/MS.As shown in Table 7, HTP, IMT, and MTCA were not detected in any of the samples tested.Although PIC was not detected in the thigh muscle; however, a small amount of PIC was detected in the breast muscle of Group 4.

| DISCUSSION
This study evaluated the safety of L-tryptophan as a feed additive in broiler chickens, with a particular emphasis on the occurrence of EMS-like symptoms.Over the course of 27 days of feed administration, no mortalities or clinical signs were observed in any of the animals.Mean body weight and body weight gain showed significant differences between the basal feed group (Group 1) and the other groups, which can be attributed to L-tryptophan deficiency in the basal feed.However, no significant differences were observed between the groups that received L-tryptophan supplementation (Groups 2, 3, and 4).
Significant differences in some parameters of the hematology analysis were observed between Group 1 and the other groups.The ratio of eosinophils and basophils in male broiler chickens from T A B L E 6 (Continued) F I G U R E 3 Histopathology analysis of male and female broiler chickens administered test substances for 27 days.H&E-stained slides of muscle fiber degeneration or necrosis (arrow head) in the breast muscle tissue of male and female broiler chickens.Note that all lesions were observed in all animals including G1. Original magnification, 200Â, scale bar: 50 μm.G1, basal feed; G2, L-tryptophan; G3, dried L-tryptophan fermentation product; G4, dried L-tryptophan fermentation product with additional impurities.
The blood coagulation test results revealed a prolongation of prothrombin time and activated partial thromboplastin time in Groups 3 and 4, irrespective of sex.However, these changes were not statistically significant compared with Group 1 for both sexes.Several animals in each group were excluded from the statistical analysis of prothrombin time due to values that fell outside the acceptable range.
The presence of out-of-range values highlights the significant differences in the mechanisms of blood coagulation between chickens and mammals (Bigland & Triantaphyllopoulos, 1960).Prolonged blood clotting time is also typically observed in lower vertebrates (birds, reptiles, and fishes) due to the lack of thromboplastic substance within their vessels (Bigland, 1964).Therefore, the occurrence of these out-of-range values was expected, given the unique characteristics of poultry serum and plasma, which differ from those of mammals.
In male chickens in Group 2, there was a slight increase in aspartate aminotransferase and alanine aminotransferase levels.
However, all changes in the liver were not considered to be related to the test substance, as there were no significant changes observed in the histopathological analysis and no consistency observed between sexes.The histopathological findings in the liver including periportal inflammatory cell infiltration, hepatocyte degeneration or necrosis, and extramedullary hematopoiesis were not considered to be test substance related, as these findings were also observed in the animals including Group 1. Minimal-to-moderate muscle fiber degeneration or necrosis was seen in the majority of the chickens, including those in Group 1.This phenomenon is commonly observed in broiler chickens, particularly after 28 days of age, and its incidence has been reported to increase with age (Radaelli et al., 2017).Thus, the muscle fiber degeneration or necrosis observed in this study are considered to be spontaneous lesions commonly found in poultry, regardless of the test substances.Additionally, the skin lesions, erosion, or ulceration observed in the epidermis, as well as the inflammation, were considered to be contact dermatitis commonly found in broiler chickens due to various factors such as seasonal conditions, farm-growing, or humidity (Dinev et al., 2019).Microbial infections in poultry can also induce contact dermatitis (Glisson, 1998;Swelum et al., 2021;Thøfner & Christensen, 2021); however, we did not observe animal mortality, necrotic enteritis, respiratory symptoms, or other diseases caused by microbial infections in this study.Therefore, it can be considered that the contact dermatitis observed is an incidental result and not caused by microbial infections.
In this study, due to the elevated absolute eosinophil counts observed in all broiler chickens, including Group 1, surpassing the diagnosis criteria for eosinophil count in humans (1000 cells/mm 3 ), it was impossible to make a direct comparison with human EMS diagnosis criteria.The male broiler chickens in Group 2 showed significantly higher eosinophil and basophil counts compared to those in Group 1.However, all differential leukocyte counts were decreased in the female broiler chickens in Groups 3 and 4. Furthermore, there was no distinct evidence of perimyositis or fasciitis in the muscle and skin based on histopathological analysis.Therefore, the changes observed in this study were considered unrelated to EMS since there was no correlation between sex and the histopathological criteria did not align with the observed hematology results.
After conducting a residue analysis on L-tryptophan impurities in the thigh and breast, which are commonly consumed as meat products, we observed that the majority of these impurities were not detected.Only a small amount of PIC was detected in the breast muscle of Group 4. Therefore, most of the ingested L-tryptophan impurities seem to be metabolized or excreted.Considering the residues of L-tryptophan impurities in broiler chickens, human consumption of L-tryptophan impurities through meat products may be negligible.Consequently, it is improbable that the residue of L-tryptophan impurities derived from feed is associated with human safety concerns.
impurities tested in this study can be used as a feed additive without concern for animal and human safety.
four animals were excluded from the statistical analysis due to the out-of-range values of prothrombin time.b n = 1; seven animals were excluded from the statistical analysis due to the out-of-range values of prothrombin time.c n = 7; one animal was excluded from the statistical analysis due to the out-of-range values of prothrombin time.*Significantdifference compared to G1 (P < 0.05).T A B L E 4 Mean biochemical parameters in male and female broiler chickens fed test substances.
two animals were excluded from the statistical analysis since no result was revealed due to the lack of serum volume.b n = 5; three animals were excluded from the statistical analysis since no result was revealed due to the lack of serum volume.c n = 7; one animal was excluded from the statistical analysis since no result was revealed due to the lack of serum volume.d n = 1; seven animals were excluded from the statistical analysis due to the out-of-range values of alkaline phosphatase.e n = 3; five animals were excluded from the statistical analysis due to the out-of-range values of alkaline phosphatase.f n = 5; three animals were excluded from the statistical analysis due to the out-of-range values of alkaline phosphatase.g n = 6; two animals were excluded from the statistical analysis due to the out-of-range values of alkaline phosphatase.h n = 7; one animal was excluded from the statistical analysis due to the out-of-range values of alkaline phosphatase.F I G U R E 1 Immunological analysis of male and female broiler chickens administered test substances for 27 days.(A) IL-4, (B) IL-5, (C) IL-6, (D) TGF-β2, (E) TGF-β3 (*P < 0.05).G1, basal feed; G2, L-tryptophan; G3, dried L-tryptophan fermentation product; G4, dried L-tryptophan fermentation product with additional impurities.n = 16, 8 males and 8 females for each group.

F
I G U R E 2 Changes in relative organ weight of male and female broiler chickens administered test substances for 27 days.(A) Liver, (B) spleen, (C) thymus, (D) heart, (E) kidney.G1, basal feed; G2, L-tryptophan; G3, dried L-tryptophan fermentation product; G4, dried L-tryptophan fermentation product with additional impurities.n = 16, 8 males and 8 females for each group.
G1, basal feed; G2, L-tryptophan; G3, dried L-tryptophan fermentation product; G4, dried L-tryptophan fermentation product with additional impurities.a n = 7; no histopathological results due to the absence of tissue sampling during necropsy.Groups 3 and 4 increased significantly, while leukocytes, including differential parameters, decreased significantly in female broiler chickens from Groups 3 and 4.However, these changes could be considered to be unrelated to the test substance because no significant changes were found in the lymphatic organs (e.g., thymus and bursa of Fabricius) upon histopathological examination.In addition, it has been reported that the range of hematologic parameters in poultry, including Ross 308, is very wide, and their changes could be attributed to physiological, environmental, and dietary conditions(Al-Nedawi, 2018;Harrison & Lightfoot, 2005;Talebi et al., 2005).
T A B L E 7 Residues of L-tryptophan impurities.